1. Field of the Invention
The invention provides nucleotide sequences encoding for the superoxide dismatase (sod) gene and a process for the fermentative preparation of nucleotides, vitamins and L-amino acids, in particular L-lysine using coryneform bacteria in which the sod gene is amplified.
2. Background Information
Nucleotides, vitamins and L-amino acids, in particular L-lysine, are used in the foodstuffs industry, in animal nutrition, in human medicine and in the pharmaceutical industry.
It is known that these substances can be prepared by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Constant efforts are made to improve the method of preparation due to the high degree of importance of these substances. Process improvements may relate to fermentation engineering factors such as e.g. stirring and supplying with oxygen, or the composition of the nutrient medium, such as e.g. the sugar concentration during fermentation, or the working up process aimed at obtaining the product itself by e.g. ion exchange chromatography or the intrinsic power of the microorganism itself.
To improve the power of the microorganisms, the methods of mutagenesis, selection and mutant choice are used. Strains which are resistant to antimetabolites or which are auxotrophic for significant regulatory intermediates are obtained in this way and produce nucleotides, vitamins and amino acids.
For some time now the methods of recombinant DNA engineering have also been used for the strain-improvement of L-amino acid-producing strains of Corynebacterium glutamicum, by amplifying individual amino acid biosynthetic genes and investigating the effect on L-amino acid production.
U.S. Pat. No. 5,179,010 describes strains of coryneform bacteria which are e.g. resistant to methylviologen or benzoyl peroxide, have an increased activity to superoxide dismutase and have an improved lysine yield. These strains were produced by non-targeted mutagenesis using the mutagen N-methyl-N-nitro-N-nitrosoguanidine. The increase in the concentration of superoxide dismutase in the strains mentioned there was at most 56%.
U.S. Pat. No. 4,529,697 describes mutants of coryneform bacteria which produce glutamic acid. The increase in the concentration of superoxide dismutase in the strains mentioned there was at most 105%.
The inventor has formulated the object as the provision of new steps for the improved handling of superoxide dismutase from coryneform bacteria. These steps can be used during the fermentative preparation of nucleotides, vitamins and L-amino acids, in particular L-lysine.
Nucleotides, vitamins and L-amino acids, in particular L-lysine, are used in the foodstuffs industry, in animal nutrition, in human medicine and in the pharmaceutical industry. Lysine-producing strains of coryneform bacteria are known from the prior art, in which the concentration of superoxide dismutase is increased by 27 to 56% and which liberate amplified lysine. These strains were obtained by non-targeted mutagenesis.
Whenever L-lysine or lysine is mentioned in the following this is intended to mean not only the base but also salts such as, for example, lysine monohydrochloride or lysine sulfate.
The invention provides a preferably recombinant DNA from the Corynebacterium source which can replicate in coryneform microorganisms and which contains at least the nucleotide sequence which encodes for the sod gene represented in SEQ-ID-No.1.
The invention also provides a replicable DNA in accordance with Claim 1 comprising:
(i) the nucleotide sequence, shown in SEQ-ID-No.1, or
(ii) at least one sequence which corresponds to the sequence (i) within the region of degeneration of the genetic code, or
(iii) at least one sequence which hybridizes with the sequence which is complementary to sequence (i) or (ii) and optionally
(iv) functionally neutral sense mutations in (i).
Coryneform microorganisms, in particular the strain Corynebacterium, transformed by the introduction of the replicatable DNA mentioned above, are also provided by the invention.
Furthermore the invention provides a process for the fermentative preparation of nucleotides, vitamins and L-amino acids, in particular L-lysine, using coryneform bacteria which in particular already produce the relevant product and in which the nucleotide sequences encoding for the sod gene are amplified, in particular are overexpressed.
The expression xe2x80x9camplificationxe2x80x9d in this connection describes the increase in the intracellular activity of one or more enzymes in a microorganism which are encoded by the corresponding DNA, for example by increasing the copy number of the gene or genes, using a strong promoter or a gene which encodes for a corresponding enzyme with high activity and optionally combining these steps.
The microorganisms which are the subject of the present invention can produce nucleotides, vitamins and L-amino acids, in particular L-lysine, from glucose, saccharose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They may be members of the coryneform bacteria family in particular the genus Corynebacterium. In the case of the Corynebacterium genus, in particular the species Corynebacterium glutamicum, it should be mentioned that this is well-known in the specialist field for its ability to produce L-amino acids.
Suitable strains of the genus Corynebacterium, in particular the species Corynebacterium glutamicum are for example the known wild-type strains
Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium ammoniagenes ATCC6871
Corynebacterium thermoaminogenes FERM BP-1539
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020
Corynebacterium melassecola ATCC17965
Brevibacterium ammoniagenes IF012072
and mutants or strains prepared therefrom which can produce nucleotides, vitamins and L-amino acids,
such as for example the 5xe2x80x2-inosinic acid-producing strains
Corynebacterium ammoniagenes ATCC15190
Corynebacterium ammoniagenes ATCC15454
Corynebacterium glutamicum ATCC14998 or
such as for example the 5xe2x80x2-guanylic acid-producing strains
Corynebacterium glutamicum ATCC21171 or
Corynebacterium ammoniagenes ATCC19216 or
such as for example the L-lysine producers
Corynebacterium glutamicum FERM-P 1709
Brevibacterium flavum FERM-P 1708
Brevibacterium lactofermentum FERM-P 1712
Corynebacterium glutamicum FERM-P 6463 and
Corynebacterium glutamicum FERM-P 6464
Corynebacterium glutamicum DSM 5714.
The inventors were able to isolate the new sod gene from Corynebacterium melassecola ATCC17965.
Here, the superoxide dismutase enzyme protein was first purified to homogeneity using chromatographic methods. Methods and instructions for protein purification and preparation are fully described e.g. in the textbook by Schleifer and Wensink: Practical Methods in Molecular Biology (Springer Verlag, Berlin, Germany, 1981), in the manual by Harris and Angal: Protein Purification Methods: A Practical approach (IRL Press, Oxford, UK, 1989), in the textbook by Scopes: Protein Purification: Principles and Practice, 3rd ed. (Springer Verlag, New York, USA, 1993) and in generally well-known textbooks and manuals. The pure enzyme protein can then be broken down into peptides by treating with suitable enzymes such as e.g. trypsin or chymotrypsin. The amino acid sequence in these peptides can be determined by the method of N-terminal sequencing described by Edman (Archives of Biochemistry 22, 475, (1949)). Methods and instructions for protein sequencing are given e.g. in Smith: Protein Sequencing Protocols: Methods in Molecular Biology, Vol. 64 and Vol. 112 (Humana Press, Totowa, N.J., USA, 1996) and in Kamp et al.: Protein Structure Analysis: Preparation, Characterization and Microsequencing (Springer Verlag, New York, N.Y., USA, 1997). The amino acid sequence in the superoxide dismutase enzyme protein can be partly or completely determined in this way, depending on the degree of complexity.
By exploiting the known use of a codon for coryneform bacteria (Malumbres et al. (Gene 134, 15-24 (1993)), synthetic oligonucleotides can be synthesized and used as primers for amplifying the corresponding chromosomal DNA segments by means of the polymerase chain reaction (PCR). Instructions for this can be found by a person skilled in the art, inter alia, for example in the manual by Gait: Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994). The DNA fragment of the sod gene obtained in this way is then cloned using known methods such as described e.g. in Sambrook et al.: Molecular Cloning: A Laboratory Manual 2nd ed. (Cold Spring Harbor Laboratory Press, USA, 1989) and can be used as probes for testing the complete gene including its 5xe2x80x2 and 3xe2x80x2 flanks in gene banks.
The construction of gene banks is described in generally well-known textbooks and manuals. The following may be mentioned as examples, the textbook by Winnacker: Gene und Klone, Eine Einfuhrung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990) or the manual by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A very well known gene bank is that of E. coli K-12 strain W3110 which was constructed by Kohara et al. (Cell 50, 495-508 (1987)) in xcex-vectors. Bathe et al. (Molecular and General Genetics, 252:255-265, 1996) describe a gene bank of C. glutamicum ATCC13032, which was constructed with the aid of the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164) in E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575). Bormann et al. (Molecular Microbiology 6(3), 317-326)) also describe a gene bank of C. glutamicum ATCC13032 using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-198 (1980)). To produce a gene bank of C. glutamicum in E. coli, plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19:259-268) may also be used. Suitable hosts are in particular those E. coli strains which are restriction and recombination defective. An example of these is the strain DH5xcex1mcr described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87, (1990) 4645-4649). The long DNA fragments cloned with the aid of cosmids can then again be sub-cloned in vectors currently used for sequencing and then sequenced as is described e.g. in Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America USA, 74:5463-5467, 1977).
The DNA sequences obtained can then be tested with known algorithms or sequence analysis programs such as e.g. that of Staden (Nucleic Acids Research 14, 217-232 (1986)), the GCG program by Butler (Methods of Biochemical Analysis 39, 74-97 (1998)) the FASTA algorithm of Pearson and Lipman (Proceedings of the National Academy of Sciences USA 85, 2444-2448 (1988)) or the BLAST algorithm of Altschul et al. (Nature Genetics 6, 119-129 (1994)) and compared with the sequence registers present in publicly accessible data banks. Publicly accessible banks for nucleotide sequences are for example those in the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany) or those in the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA).
The new DNA sequences of C. glutamicum encoding for the sod gene which was obtained in this way is a constituent of the present invention as SEQ ID NO 1. Furthermore the amino sequence of the corresponding protein was derived from the DNA sequence present. The amino acid sequence of the sod gene product is represented in SEQ ID NO 2.
Encoding DNA sequence which was produced from SEQ ID NO 1 by the degenerability of the genetic code is also a constituent of the invention. In the same way DNA sequences which hybridize with SEQ ID NO 1 or parts of SEQ ID NO 1 are also a constituent of the invention. In the specialist field, furthermore, conservative amino acid exchanges such as e.g. the exchange of glycine for alanine or of aspartic acid for glutamic acid in proteins, as xe2x80x9csense mutationsxe2x80x9d, are also known and do not lead to any basic modification in the activity of the protein i.e. they are functionally neutral. Furthermore, it is known that changes to the N and/or C terminus of a protein cannot substantially impair or even stabilize its function. Data relating to this can be found by a person skilled in the art, inter alia, in Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), in O""Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) and in well-known textbooks on genetics and molecular biology. Amino acid sequences which are produced in a corresponding manner from SEQ ID NO 2 are also a constituent of the invention.
Amplification of the sod gene in coryneform bacteria leads to an unusually high increase in the superoxide dismutase concentration in the microorganism.
To produce an overexpression, the copy number of the corresponding gene can be increased, or the promoter and regulation region or the ribosome bonding site, which is located upstream of the structure gene, can be mutated. Expression cassettes, which are incorporated upstream of the structure gene, operate in the same way. It is also possible to increase expression during the course of fermentative L-lysine production with inducible promoters. Expression is also improved by measures aimed at prolonging the lifetime of m-RNA. Furthermore, enzyme activity can also be amplified by inhibiting degradation of the enzyme protein. The genes or gene constructs can either be present in plasmids with different copy numbers or be integrated and amplified in the chromosome.
Alternatively, overexpression of the relevant genes may also be achieved by modifying the composition of the media and management of the culture.
Instructions for these procedures may be found by a person skilled in the art, inter alia, in Martin et al. (Bio/Technology 5, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in European patent EP-B-0 472 869, in U.S. Pat. No. 4,601,893, in Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991)), in Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), in Labarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in patent application WO 96/15246, in Malumbres et al. (Gene 134, 15-24 (1993)), in the Japanese patent document JP-A-10-229891, in Jensen and Hammer (Biotechnology and Bioengineering, 58, 191-195 (1998)), in Makrides (Microbiological Reviews, 60:512-538 (1996)) and in well-known textbooks on genetics and molecular biology.
An example of a plasmid with the help of which the sod gene can be overexpressed is pMM23 (FIG. 1) which is contained in the strain MH20-22B/pMM23. Plasmid pMM23 is an E. coli-C. glutamicum shuttle vector based on plasmid pBL1 (Ferandez-Gonzalez et al., Journal of Bacteriology 176(11), 3154-3161 (1994)), pACYC184 (Chang and Cohen, Journal of Bacteriology 134(3), 1141-1156 (1978)) and the trc promoter (Brosius et al. , Journal of Biological Chemistry 260, 3539-3541 (1985)) which carries the sod gene. Other plasmid vectors which can be replicated in C. glutamicum such as e.g. pEKEx1 (Eikmanns et al., Gene 102: 93-98 (1991)) or pZ8-1 (European patent 0 375 889) may be used in the same way as starting vectors for cloning and expressing the sod gene.
It may also be advantageous for the production of nucleotides, vitamins and in particular L-amino acids to overexpress one or more enzymes in the particular biosynthetic pathway in addition to the sod gene.
Thus, for example, for the preparation of nucleotides
the purF gene encoding for the glutamine PRPP-amidotransferase may be simultaneously overexpressed
the carAB gene encoding for carbamoyl phosphate synthetase may be simultaneously overexpressed.
Thus, for example, for the preparation of D-pantothenic acid
the panD gene encoding for aspartate decarboxylase (Dusch et al., Applied and Environmental Microbiology 65, 1530-1539 (1999)) may be simultaneously overexpressed.
Finally, for example, for the preparation of L-lysine
the dapA gene encoding for dihydrodipicolinate synthase may be simultaneously overexpressed (EP-B 0 197 335), or
a DNA fragment promoting S-(2-aminoethyl)-cysteine resistance may be simultaneously amplified (EP-A 0 088 166).
Furthermore it may be advantageous for the production of nucleotides, vitamins and in particular L-amino acids, quite particularly L-lysine, to switch off undesired side reactions apart from overexpression of the sod gene (Nakayama: xe2x80x9cBreeding of Amino Acid Producing Micro-organismsxe2x80x9d, in Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.) Academic Press, London, UK, 1982).
The microorganisms prepared according to the invention may be cultivated continuously or batchwise in a batch process or a fed batch process or a repeated fed batch process for the purposes of producing metabolic products. A review of known cultivation methods is given in the textbook by Chmiel (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
The culture medium to be used must satisfy the requirements of the particular strains in an appropriate manner. Descriptions of culture media for various microorganisms are given in the book xe2x80x9cManual of Methods for General Bacteriologyxe2x80x9d by the American Society for Bacteriology (Washington D.C., USA, 1981). Sugar and carbohydrates such as e.g. glucose, saccharose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as e.g. soya oil, sunflower oil, peanut oil and coconut fat, fatty acids such as e.g. palmitic acid, stearic acid and linoleic acid, alcohols such as e.g. glycerol and ethanol and organic acids such as e.g. acetic acid can be used as sources of carbon. These substances may be used individually or as a mixture. Organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, maize steep water, soya bean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate may be used as sources of nitrogen. The sources of nitrogen may be used individually or as a mixture. Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts may be used as sources of phosphorus. The culture medium must also contain salts of metals such as e.g. magnesium sulfate or iron sulfate which are required for growth. Finally essential growth substances such as amino acids and vitamins may also be used in addition to the substances mentioned above. Over and above these, appropriate precursors may also be added to the culture medium. The feedstocks mentioned may be added to the culture as a one-off batch or may be supplied during cultivation in an appropriate manner.
Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acid compounds such as phosphoric acid or sulfuric acid may be used in an appropriate manner to control the pH of the culture. Anti-foam agents such as e.g. fatty acid polyglycol esters may be used to control the production of foam. To maintain the stability of plasmids, suitable selectively acting substances e.g. antibiotics may be added to the medium. In order to maintain aerobic conditions, oxygen or oxygen-containing gas mixtures such as e.g. air may be introduced into the culture. The temperature of the culture is generally 20xc2x0 C. to 45xc2x0 C. and preferably 25xc2x0 C. to 40xc2x0 C. Cultivation is continued until a maximum in the desired L-amino acid has been produced. This objective is normally achieved within 10 hours to 160 hours.
The following microorganisms were deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ, Mascheroder Weg 1b, D-38124, Braunschweig, Germany) on Jun. 8, 1999 and in accordance with the Budapest treaty:
Esherichia coli strain XL1 blue/pMM23 as DSM 12860.
Corynebacterium melassecola strain 1019 as DSM 12859.
The process according to the invention is used for the fermentative preparation of nucleotides, vitamins and in particular L-amino acids with coryneform bacteria, quite particularly the preparation of L-lysine.